Leukocytes entering an inflammatory site migrate between tightly apposed endothelial cells (EC) into the underlying tissues, a process called transendothelial migration (TEM). My long-term objectives are to understand, on a molecular level, what controls inflammation in general and (TEM). My long-term objectives are to understand, on a molecular level, what controls inflammation in general and TEM in particular. This proposal centers on PECAM, an adhesion molecule on the surfaces of leukocytes and concentrated in the junctions between EC. The studies funded by the original grant allowed me to study PECAM and characterize the several adhesive interactions it can participate in. The most significant of these is TEM, where it is required for the TEM of most monocytes and neutrophils. In the studies we will perform over the next years, we will characterize two distinct steps in leukocyte emigration in which PECAM participates: TEM and migration of leukocytes into the interstitium. Each appears to be mediated independently by distinct portions of the PECAM molecule. Our preliminary data come from studies performed using monoclonal antibodies, whose domains on PECAM are known, to block PECAM function, In order to more precisely define the regions of the PECAM molecular that participate in these functions, PECAM-Ig chimeras are being made. These are comprised of full-length r modified PECAM fused to the IgG heavy chain. The Ig heavy chains facilitate quantitation of binding and purification of these proteins and also provide the opportunity to study the role of valency in PECAM action. PECAM-Ig chimeras will be used to identify the domains of PECAM that function in TEM on both monocytes and neutrophils as well as on EC. In addition the domains that are responsible for PECAM- dependent movement into the interstitium will be identified. Immunoelectron microscopy studies are planned to analyze PECAM and TEM at the ultrastructural level. These studies will examine how PECAM is disposed along the EC borders and the leukocyte pseudopod and whether these change during TEM. This should shed light on some potential mechanisms by which PECAM functions. Perhaps most important, our in vitro results will be tested in several in vivo models selected to provide us with unique insights into PECAM function. The role of PECAM will be examined when models of CD18-dependent and CD18-independent inflammation are compared. Murine PECAM-Ig chimeras are being made to parallel the in vitro studies. Intraviral microscopy will demonstrate the actions of anti-PECAM reagents in ongoing inflammation. We will study TEM itself and migration through the interstitium of the mesentery.